EP2284210A1 - Polyplex ternaire à conversion de charge - Google Patents

Polyplex ternaire à conversion de charge Download PDF

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Publication number
EP2284210A1
EP2284210A1 EP09738905A EP09738905A EP2284210A1 EP 2284210 A1 EP2284210 A1 EP 2284210A1 EP 09738905 A EP09738905 A EP 09738905A EP 09738905 A EP09738905 A EP 09738905A EP 2284210 A1 EP2284210 A1 EP 2284210A1
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Prior art keywords
group
polyplex
integer
pasp
det
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EP2284210B1 (fr
EP2284210A4 (fr
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Kazunori Kataoka
Yan Lee
Nobuhiro Nishiyama
Kanjiro Miyata
Makoto Oba
Shigehiro Hiki
Mai Sanjo
Hyunjin Kim
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University of Tokyo NUC
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/40—Polyamides containing oxygen in the form of ether groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to a polymer complex (polyplex) containing a nucleic acid for use as a nonviral synthetic vector capable of delivering the nucleic acid to a target cell. More particularly, the present invention relates to a charge conversional ternary polyplex comprising a nucleic acid, a cationic polymer and an anionic polymer, wherein the anionic polymer covers the surface of a complex comprising the cationic polymer and the nucleic acid.
  • DNA or RNA delivery to a target cell mediated by a nonviral synthetic vector has been widely recognized as a promising alternative method for delivery that uses a viral vector which has been confronting safety issues specific to biological properties [1] . Nonetheless, even in the case of a nonviral vector, the main concern is the conflict between the delivery efficiency and the safety issues (in particular, chemical toxicity). While most vectors having high transfection efficiency show high toxicity, vectors with low toxicity are often associated with low transfection efficiency. Behr et al. introduced a concept of endosomal escape through "proton-sponge" effect, a hypothesis proposed for polyethylenimine (PEI), into the field of gene delivery.
  • PEI polyethylenimine
  • polyplexes are used only for limited applications [2] .
  • One major reason for limited development of polycation is presumably that different functions, which may be opposing functions, of the polyplex are required at different stages of the delivery process.
  • a moiety having a high amine density in a polyplex is important to overcome the endosomal membrane barrier since a protonated potential of the polyplex could be a cause of endosome buffering and membrane destabilization [3] .
  • the present inventors have examined a new approach for a polyplex design that exerts both properties of high transfection activity and low toxicity by integrating a charge conversional moiety into a polyplex structure.
  • maleic acid amide derivatives i.e., cis-aconitic acid amide and citraconic acid amide
  • cis-aconitic acid amide and citraconic acid amide bear negative charges at neutral pH, they are rapidly degraded at weak acidity of pH 5.5 and expose the positively-charged amine [8] .
  • a ternary polyplex (a polyplex having pDNA/polycation/polyanion having degrading side-chain) formed by the present inventors by covering the surface of a positively-charged polyplex with a polymer derived from degrading amide, by which the ternary polymer maintains neutrally- to negatively-charged properties outside the cell.
  • the charge conversional moieties are expected to charge positively and promote endosomal escape of the polyplex owing to membrane disruption. This scheme is illustrated in Figure 1 .
  • the present invention has an objective of providing a polymer that is negatively charged outside the cell but that undergoes charge conversion and positively charged once entering the endosome.
  • the present invention also has an objective of providing a complex (polyplex) comprising the polymer, namely a ternary polyplex which overcomes the problem (dilemma) existing between gene transfection efficiency and safety and which can fulfill low toxicity and high gene transfection efficiency.
  • the present inventors have gone through keen examination. As a result, they found that a polymer obtained by causing a polycation to react with citraconic anhydride or cis-aconitic anhydride is negatively charged under a neutral condition but its charge is converted to give a cation under an acidic condition, and therefore the polymer shows low toxicity and high gene transfection efficiency, thereby accomplishing the present invention.
  • the present invention is an anionic polymer represented by the following Formula (1).
  • R 11 and R 12 independently represent a hydrogen atom or an optionally substituted linear or branched alkyl group with a carbon number of 1-12
  • R 13 represents a conjugate of a residue derived from an amine compound having a primary amine
  • R a and R b independently represent a hydrogen atom, or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group, heterocyclic alkyl group, hydroxy group, alkoxy group or aryloxy group
  • R a and R b may bind to each other to form an aromatic ring or a cycloalkyl ring with the carbon atoms to which they are each bound, where the binding between the carbon atoms to which R a and R b are each bound is either a single bond
  • examples of the residue derived from an amine compound having a primary amine include: a group represented by General Formula (11) below: -NH-(CH 2 ) r -X 11 (11) (wherein, X 11 represents an amine compound residue derived from primary, secondary or tertiary amine compound or quaternary ammonium salt, and r represents an integer of 0-5); and a group represented by General Formula (12) below: -[NH-(CH 2 ) s1 ] t1 -X 12 (12) (wherein, X 12 is synonymous with X 11 , and s1 and t1, independently from each other and independently between [NH-(CH 2 ) s1 ] units, represent integers of 1-5 and 2-5, respectively), and preferably a group represented by -NH-NH 2 or -NH-(CH 2 ) 2 -NH-(CH 2 ) 2 -NH 2 .
  • the compound represented by Formula (I) above may be, for example, at least one type of the compounds represented by Formulae (Ia)-(Ig) below.
  • the present invention also provides a polymer complex comprising a nucleic acid, a cationic polymer and the above-mentioned anionic polymer.
  • examples of the nucleic acid include plasmid DNA and siRNA.
  • cationic polymer examples include any compound selected from the group consisting of:
  • the present invention also provides a device or a kit for delivering a nucleic acid into a cell, comprising the polymer complex.
  • an anionic polymer covers the surface of a complex of a cationic polymer and a nucleic acid.
  • the anionic polymer can change its charge from negative at neutral pH to positive at weak acidic pH.
  • a specific example of such an anionic polymer includes a polymer (specifically, "pAsp (DET-Aco)") shown in Figure 2A(c) .
  • the present invention can provide a charge conversional ternary polyplex that is charged negatively under a neutral condition but its charge is converted to give a cation under an acidic condition.
  • a polyplex of the present invention is capable of delivering a nucleic acid such as DNA or siRNA to such types of cells in a highly efficient manner without evoking toxicity. Medically speaking, major drawbacks of nucleic acid delivery include low efficiency, high toxicity and instability in the blood.
  • the charge conversional ternary polyplex of the present invention has very low toxicity since it is negatively charged outside the cell and thus is extremely useful as a gene vector in vivo.
  • the present invention is a polymer complex (polyplex) comprising a nucleic acid, a cationic polymer and an anionic polymer, wherein the anionic polymer is negatively charged at neutral pH and its charge is altered to positive at weak acidic pH.
  • the present invention also provides an anionic polymer that constitutes the above-mentioned polymer complex.
  • An anionic polymer of the present invention is represented by the following Formula (1) and is used as a charge conversional polymer.
  • R 11 and R 12 independently represent a hydrogen atom or an optionally substituted linear or branched alkyl group with a carbon number of 1-12
  • R 13 represents a conjugate of a residue derived from an amine compound having a primary amine and a compound represented by Formula (I) below:
  • R a and R b independently represent a hydrogen atom, or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group, heterocyclic alkyl group, hydroxy group, alkoxy group or aryloxy group
  • R a and R b may bind to each other to form an aromatic ring or a cycloalkyl ring with the carbon atoms to which they are bound, where the binding between the carbon atoms to which R a and R b are
  • examples of the residue derived from an amine compound having a primary amine include groups represented by General Formulae (11) or (12) below.
  • -NH-(CH 2 ) r -X 11 (11) (wherein, X 11 represents an amine compound residue derived from primary, secondary or tertiary amine compound or quaternary ammonium salt, and r represents an integer of 0-5) -[NH-(CH 2 ) s1 ] t1 -X 12 (12)
  • X 12 is synonymous with X 11 , and s1 and t1, independently from each other and independently between [NH-(CH 2 ) s1 ] units, represent integers of 1-5 and 2-5, respectively).
  • a residue derived from an amine compound having a primary amine is preferably one represented by -NH-NH 2 or -NH-(CH 2 ) 2 -NH-(CH 2 ) 2 -NH 2 .
  • the compound represented by Formula (I) above may be, for example, at least one type of the compounds represented by Formulae (Ia)-(Ig) below.
  • a preferable example of an anionic polymer contained in the polyplex of the present invention includes pAsp(DET-Aco), where the structure of pAsp(DET-Aco) is a polymer having an anionic side-chain by binding an N'-cis-aconityl group to an amino group at the end of the side-chain of a cationic polymer pAsp(DET) (that is, an amino acid polymer obtained by binding an ethylenediamine unit to an aspartate backbone).
  • pAsp(DET-Aco) may be synthesized by causing a primary amino group of a hydrophobic group (e.g., PLL) to react with a compound represented by Formula I (for example, cis-aconityl anhydride).
  • a hydrophobic group e.g., PLL
  • Formula I for example, cis-aconityl anhydride
  • Binding between a residue derived from an amine compound having a primary amine and a compound represented by Formula (I) or a derivative thereof may be obtained, for example, by binding (covalently linking) the compound represented by Formula (I) above and the amino group of the amine compound to give a structure represented by Formula (I') below.
  • a derivative refers to any compound derived from a compound having General Formula (I) above as a fundamental backbone.
  • such a derivative may be a compound obtained by replacing COOH of compound (Ic) with an alkyl group, a compound obtained by replacing a methyl group of compound (Ib) or (Ie) with other alkyl group, or a compound obtained by replacing an aromatic ring or a cycloalkyl ring with at least one halogen atom.
  • a substituent group may be a saturated or unsaturated acyclic or cyclic hydrocarbon group.
  • an acyclic hydrocarbon group it may be either linear or branched.
  • the hydrocarbon group include a C 1 -C 20 alkyl group, a C 2 -C 20 alkenyl group, a C 4 -C 20 cycloalkyl group, a C 6 -C 18 aryl group, a C 6 -C 20 aralkyl group, a C 1 -C 20 alkoxy group and a C 6 -C 18 aryloxy group.
  • a specific cationic polymer as a constituent of a complex of the present invention is a cationic polymer having at least partially a polycation moiety.
  • a cationic polymer is not limited to the above-mentioned pAsp(DET) (comprising pAsp(DET), i.e., a fundamental backbone of pAsp(DET-Aco) above).
  • pAsp(DET) comprising pAsp(DET), i.e., a fundamental backbone of pAsp(DET-Aco) above.
  • Any polypeptides that are known to have a cationic group in the side-chain may be comprised. These may be used for carrying out the present invention in the same manner as pAsp(DET) by technical knowledge common for those skilled in the art.
  • the cationic group used herein is not limited to a group which has already been rendered cationic with a coordinated hydrogen ion but it may also comprise a group that will be cationic once it gains a hydrogen ion.
  • a polypeptide having a cationic group in the side-chain comprises polypeptides obtained through peptide bond of known amino acids having basic side-chains (lysine, arginine, histidine, etc.) as well as polypeptides obtained through peptide bond of any amino acid and subsequent substitution in the side-chain to have a cationic group.
  • Examples of a cationic polymer include compounds represented by the following Formulae (2)-(4): [wherein, R 21 and R 22 independently represent a hydrogen atom or an optionally substituted linear or branched alkyl group with a carbon number of 1-12, R 23 represents a residue derived from an amine compound having a primary amine, m3 represents an integer of 10-500, and m4 represents an integer of 1-5]; [wherein, R 31 and R 32 are synonymous with R 21 and R 22 , respectively, R 33 represents an optionally substituted saturated or unsaturated linear or branched aliphatic hydrocarbon group or steroloxycarbonyl group with a carbon number of 11-27, m5 and m6 independently represent an integer of 0-500 (provided that the sum of m5 and m6 is an integer of 10-500), m7 represents an integer of 1-5, m8 represents an integer of 1-5, and the sign "/" indicates that the sequential order of the (m5 + m6) numbers of monomer units on
  • R 2 containing a cationic group represents a residue derived from an amine compound having a primary amine.
  • Examples of -R 23 group include groups represented by General Formulae (22) and (23) below, among which a group represented by General Formula (23) below is preferable: -NH-(CH 2 ) r -X 21 (22) [wherein, X 21 represents a primary, secondary or tertiary amine compound or an amine compound residue derived from quaternary ammonium salt, and r represents an integer of 0-5]; and -[NH-(CH 2 ) s2 ] t2 -X 22 (23) [wherein, X 22 represents a primary, secondary or tertiary amine compound or an amine compound residue derived from quaternary ammonium salt, and s2 and t2, independently from each other and independently between [NH-(CH 2 ) s2 ] units, represent integers of 1-5 (preferably 2) and 2-5 (preferably 2), respectively].
  • -X 21 and - X 22 groups (amine compound residues) at the terminals include -NH 2 , -NH-CH 3 , -N(CH 3 ) 2 and groups represented by Formula (i)-(viii) below, among which -NH 2 is particularly preferable.
  • examples of Y include a hydrogen atom, an alkyl group (with a carbon number of 1-6) and an aminoalkyl group (with a carbon number of 1-6).
  • -R 23 in General Formula (2) is particularly preferably "-NH-NH 2 " or "-NH-(CH 2 ) 2 -NH-(CH 2 ) 2 -NH 2 ", among which the latter containing an ethylenediamine unit is more preferable.
  • m3 represents, but not limited to, an integer of 100-500, preferably 30-150 (more preferably 60-100), and m4 preferably represents, but not limited to, an integer of 1-5 (more preferably 1-2).
  • a saturated aliphatic hydrocarbon residue is equivalent to an alkyl group with a carbon number of 11-27, examples including, in addition to the above-mentioned alkyl group, a pentacosyl group, a hexacosyl group and a heptacosyl group.
  • An unsaturated aliphatic hydrocarbon residue refers to a group in which 1-5 carbon-carbon single bonds in the chain of the alkyl group are replaced by carbon-carbon double bonds.
  • Examples of an acyl group (RCO-) having such a residue (R) include, but not limited to, lauric acid (or dodecanoic acid), myristic acid (or tetradecanoic acid), palmitic acid (or hexadecane acid), palmitoleic acid (or 9-hexadecenoic acid), stearic acid (or octadecanoic acid), oleic acid, linoleic acid, linolenic acid, eleostearic acid (or 9,11,13-octadecatrienoic acid), arachidic acid, arachidonic acid, behenic acid, lignoceric acid, nervonic acid, cerotic acid and montanic acid.
  • lauric acid or dodecanoic acid
  • myristic acid or tetradecanoic acid
  • palmitic acid or hexadecane acid
  • palmitoleic acid or 9-hexadecenoic
  • sterol refers to a natural, semisynthetic or synthetic compound based on a cyclopentanone hydrophenanthrene ring (C17H28), and further refers to derivatives thereof.
  • natural sterols may be, but not limited to, cholesterol, cholestanol, dihydrocholesterol, cholic acid, campesterol and cystosterol while semisynthetic or synthetic compounds thereof may be, for example, synthetic precursors of these natural products (comprising compounds in which, if necessary, a certain functional group or a part or all of hydroxy groups, if any, may be protected with a hydroxy protecting group known in the art, or a carboxyl group is protected by carboxyl protection).
  • a sterol derivative may have a C1-12 alkyl group or a halogen atom such as chlorine, bromine or fluorine introduced into a cyclopentanone hydrophenanthrene ring to an extent that does not cause harmful effects on the purpose of the present invention, where this ring system may be saturated or partially unsaturated.
  • a residue of a sterol derivative is a group obtained by removing a hydrogen atom from a hydroxy group at position 3 in cholesterol, cholestanol or dihydroxycholesterol. More preferably, it is a group obtained by removing a hydrogen atom from a hydroxy group at position 3 in cholesterol.
  • Sterol of a steroloxycarbonyl group may be derived from animal or plant oil resources, such as cholesterol, cholestanol, dihydrocholesterol, cholic acid, campesterol and cystosterol.
  • the above-mentioned cationic polymer may be produced as follows.
  • Poly(amino acid)s represented by Formulae (2)-(4) above may be produced, for example, by: introducing a polyamine residue into the side-chain of the poly(amino acid) through aminolysis of polyamino acid ester produced by polymerization of a known aspartate ester-derived N-carbonic anhydride by using a polyamine corresponding to the polyamine residue of the groups represented by R 23 , R 33 and R 44 ; activating, if necessary, an amino group of the introduced polyamine moiety and a carboxyl group of carboxylic acid corresponding to an acyl group having the above-mentioned aliphatic hydrocarbon residue; and subsequently causing an appropriate amount of the activated carboxylic acid to react with the amino group.
  • the cationic polymer may be a polymer (homopolymer) consisting only of a polycation moiety as described above, it may be, without limitation, for example, a block copolymer or a graft polymer having a polyethylene glycol (PEG) moiety and a polycation moiety.
  • PEG polyethylene glycol
  • An appropriate and preferable embodiment may be selected according to the application of the complex of the present invention.
  • the structures (e.g., polymerization degree, etc.) of PEG and a polycation are not limited and any structure may be selected, among which the polycation is preferably a polypeptide having a cationic group in the side-chain.
  • a polyplex of the present invention may be a core-shell micellar complex which is obtained by interaction between a nucleic acid and a part (a polycation moiety) of the block copolymer that gives a core moiety as an ion complex, and the rest part (a moiety containing the PEG moiety) of the block copolymer forms a shell moiety around the core moiety.
  • block copolymers include those represented by General Formula (5) below.
  • R 51 and R 52 are synonymous with R 21 and R 22 , respectively, and independently represent a hydrogen atom or an optionally substituted linear or branched alkyl group with a carbon number of 1-12
  • R 53 and R 54 are synonymous with R 23 and represent a residue derived from an amine compound having a primary amine
  • L 1 represents NH, CO
  • L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO
  • L 3a represents NH or CO
  • q1 represents an integer of 1-6
  • m13, m14 and m15 independently represent an integer of 1-6
  • -R 53 and/or -R 54 group represents a residue derived from an amine compound having a primary amine, and may be applied in the same manner as described for General Formula (2) above.
  • it may be a group represented by General Formula (11) or (12) below, and preferably a group represented by General Formula (12).
  • the block moiety accompanied by the repeating unit number (degree of polymerization) "n” is a PEG moiety
  • the block moiety including the moieties accompanied by the repeating unit numbers "m13", “m14” and “m15” are a polycation moiety.
  • the sign "/" in the structural formula of the polycation moiety indicates that the sequential order of the monomer units on both sides of the sign is arbitrary.
  • Examples of the above-mentioned linear or branched alkyl group with a carbon number of 1-12 include a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, a sec-butyl group, a tert-butyl group, a n-pentyl group, a n-hexyl group, a decyl group and an undecyl group.
  • Examples of the above-mentioned substituent groups of the alkyl group include an acetalized formyl group, a cyano group, a formyl group, a carboxyl group, an amino group, an alkoxycarbonyl group with a carbon number of 1-6, an acylamide group with a carbon number of 2-7, a siloxy group, a silylamino group and a trialkylsiloxy group (alkylsiloxy groups independently have a carbon number of 1-6).
  • substituent group When the substituent group is an acetalized formyl group, it may be converted into another substituent group, i.e., formyl group (an aldehyde group; -CHO) by hydrolysis under mild acidic conditions.
  • formyl group an aldehyde group; -CHO
  • L 1 as a linker moiety represents NH, CO, a group represented by General Formula (13) below: -(CH 2 ) p1 -NH- (13) [wherein, p1 represents an integer of 1-6 (preferably 2-3)], or a group represented by General Formula (14) below: -L 2a -(CH 2 ) q1 -L 3a - (14) [wherein, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, L 3a represents NH or CO, and q1 represents an integer of 1-6 (preferably 2-3)].
  • n represents the number of the repeating unit (degree of polymerization) of the PEG moiety, which is specifically an integer of 0-500 (preferably 100-400, and more preferably 200-300).
  • m13, m14 and m15 as degrees of polymerization of respective monomer units constituting the polycation moiety independently represent an integer of 0-500 (preferably 0-100, more preferably 10-100, still more preferably 20-100, and yet still more preferably 30-100) (provided that the sum of m13, m14 and m15 (m13+m14+m15) is 10-500 (preferably 10-300, more preferably 20-200, still preferably 30-150, still more preferably 40-120, yet still more preferably 50-100, and particularly preferably 60-70)).
  • a molecular weight (Mw) of the cationic polymer represented by General Formula (5) is preferably, but not limited to, 23,000-45,000, and more preferably 28,000-34,000.
  • a molecular weight (Mw) of the PEG moiety is preferably 8,000-15,000, and more preferably 10,000-12,000 while a molecular weight (Mw) of the polycation moiety is preferably 15,000-30,000, and more preferably 18,000-22,000 in total.
  • a method for producing a cationic polymer represented by General Formula (5) may be, for example, but limited to, a method comprising: synthesizing a segment (a PEG segment) containing R 51 and a block moiety of a PEG chain in advance; sequentially polymerizing certain monomers on one end of this PEG segment (the end opposite to R 51 ); and, if necessary, thereafter substituting or converting the side-chain to contain a cationic group.
  • another example may be a method comprising: synthesizing the PEG segment and a block moiety containing a side-chain having a cationic group; and linking them to each other. Methods and conditions for reactions employed in these production methods may appropriately be selected or determined considering a common procedure.
  • the above-mentioned PEG segment may be prepared, for example, by employing a method for producing a PEG segment moiety of a block copolymer described in publications WO 96/32434 , WO 96/33233 and WO 97/06202 .
  • the end opposite to the -R 51 group corresponds to "-L 1 " moiety in General Formula (5), and it is preferably -NH 2 , -COOH, a group represented by General Formula (6) below: -(CH 2 ) p2 -NH 2 (6) [wherein, p2 represents an integer of 1-5 (preferably 2-3)], or a group represented by General Formula (7): -L 2b -(CH 2 ) q2 -L 3b (7) [wherein, L 2b represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, L 3b represents NH 2 or COOH and q2 represents an integer of 1-6 (preferably 2-3)].
  • a specific method for producing a cationic polymer represented by General Formula (5) may comprise: synthesizing a block copolymer by polymerizing the amino terminal with N-carbonic anhydride (NCA) of a protected amino acid such as ⁇ -benzyl-L-aspartate (BLA) and N ⁇ -Z-L-lysine by using a PEG segment derivative having the amino group at the terminal; and thereafter substituting or converting the side-chain of each block moiety with diethylenetriamine (DET) or the like to have the above-described cationic group.
  • NCA N-carbonic anhydride
  • BLA ⁇ -benzyl-L-aspartate
  • DET diethylenetriamine
  • a nucleic acid comprised as a constituent of a core moiety in a complex of the present invention is plasmid DNA or siRNA (small interfering RNA).
  • siRNA is capable of suppressing the expression of a gene of interest through RNA interference (RNAi).
  • genes of interest include, but not limited to, a cancer (tumor) gene, an antiapoptotic gene, a cell cycle-related gene and a proliferation signal gene.
  • the base length of siRNA is not limited but generally less than 30 bases (preferably 19-21 bases).
  • the nucleic acid comprised is not limited to plasmid DNA and siRNA only, and if necessary, other nucleic acids such as antisense Oligo DNA, decoy nucleic acid (double-stranded DNA), various RNAs, various suppressor genes (cancer suppressor gene, etc.), a chemically-modified nucleic acid or a non-native nucleic acid analog (for example, PNA (peptide nucleic acid)) may be comprised.
  • nucleic acid such as antisense Oligo DNA, decoy nucleic acid (double-stranded DNA), various RNAs, various suppressor genes (cancer suppressor gene, etc.), a chemically-modified nucleic acid or a non-native nucleic acid analog (for example, PNA (peptide nucleic acid)) may be comprised.
  • PNA peptide nucleic acid
  • nucleic acid molecule such as siRNA is a polyanion, it may bind to (associate with) the side-chain of the polycation moiety of the above-described block copolymer through electrostatic interaction.
  • a complex of the present invention is a complex of a polymer compound that can be obtained by forming a polyplex with a nucleic acid and a polycation and adding a charge conversional polymer, i.e., the above-mentioned anionic polymer (for example, pAsp (DET-Aco)), thereto.
  • a charge conversional polymer i.e., the above-mentioned anionic polymer (for example, pAsp (DET-Aco)
  • This complex is also referred to as a ternary polyplex (polyion complex: PIC).
  • a ternary polyplex of the present invention comprises a core moiety consisting of a nucleic acid and a polycation moiety bound via electrostatic interaction, and an anionic polymer (for example, pAsp(DET-Aco)) surrounding the core moiety.
  • an anionic polymer for example, pAsp(DET-Aco)
  • a ternary polyplex of the present invention may readily be prepared, for example, by mixing a nucleic acid and a cationic polymer in any buffer (for example, Tris buffer) to form a polyplex and subsequently adding pAsp(DET-Aco) thereto.
  • any buffer for example, Tris buffer
  • the size of the PIC of the present invention is not limited but, for example, the particle size as measured by dynamic light scattering measurement (DLS) is preferably 5-200 nm, and more preferably 10-150 nm.
  • DLS dynamic light scattering measurement
  • An example of the ternary polyplex of the present invention includes a conjugate in which a nucleic acid is bound to a polycation moiety in a block copolymer as the above-described cationic polymer via electrostatic interaction.
  • the above-described cationic polymer further contains a polyethylene glycol moiety such that the nucleic acid and the polycation moiety of the cationic polymer form a core moiety, and the polyethylene glycol moiety of the cationic polymer forms a shell moiety around the core moiety.
  • the ternary polyplex of the present invention may readily be prepared by mixing a nucleic acid (for example, plasmid DNA or siRNA) with a block copolymer in any buffer (for example, Tris buffer, etc.), but it should be prepared under sufficiently reductive conditions so as to avoid disulfide binding and resulting aggregation of only block copolymers prior to electrostatic binding between the block copolymer and the nucleic acid.
  • reductive conditions may be adjusted by adding DTT (dithiothreitol) or the like.
  • a mixture ratio of the block copolymer to the nucleic acid is not limited but, for example, the ratio (N/P ratio) of the total number (N) of cationic groups (for example, amino groups) in the block copolymer to the total number (P) of phosphate groups in the nucleic acid is preferably 0.5-100, more preferably 0.5-10, still more preferably 1-10, yet still more preferably 1-4, and particularly preferably 1-2.
  • the N/P ratio where the block copolymer is a copolymer represented by General Formula (5) is preferably 0.5-10, more preferably 0.5-4, and still more preferably 1-2.
  • N in this case is the sum of primary and secondary amines contained in the side-chain of the polycation moiety.
  • N refers to a group that is capable of forming an ion bond with the phosphate group in the nucleic acid through electrostatic interaction.
  • the size of the ternary polyplex of the present invention is not limited but, for example, the particle size as measured by dynamic light scattering measurement (DLS) is preferably 30-150nm, and more preferably 50-100nm.
  • DLS dynamic light scattering measurement
  • the ternary polyplex of the present invention is negatively charged in a buffer under neutral environment (pH 7.0-7.4, preferably pH 7.4), it is converted to have positive charge in a buffer under acidic environment (pH 5.0-6.8, preferably pH 5.5).
  • the extracellular pH is 7.4 and the ternary polyplex is stable in the presence of serum protein.
  • pH is reduced to 5.5 and the compound represented by Formula (I) (preferably cis-aconitic acid amide or citraconic acid amide) is degraded, where a polycation (pAsp(DET), etc.) having a function of rendering an endosomal membrane weak is exposed on the polyplex surface.
  • a polycation pAsp(DET), etc.
  • the charge conversional ternary polyplex exhibits low toxicity and high gene transfection efficiency even for a very sensitive cell such as HUVEC (human umbilical vein endothelial cell) or the like.
  • the present invention provides a nucleic acid delivery device comprising the above-described ternary polyplex (often simply referred to as a "polyplex").
  • the delivery device of the present invention can easily render a nucleic acid stable, which has been difficult to be delivered to a target cell in a stable state. Furthermore, change in the pH inside and outside the cell is utilized as means for efficiently introducing the intended nucleic acid in the core moiety of the polyplex into a target cell either in vitro or in vivo.
  • a solution containing a polyplex containing an intended plasmid or siRNA is administered to a test animal for intake into a target cell in the body. Later, the polyplex taken into the cell migrates from the endosome to the cytoplasm.
  • this binding is broken, as a result of which substitution between the contained nucleic acid and the polyanion present in the cell is promoted, where disassociation of the polyplex allows release of the intended nucleic acid into the cytoplasm.
  • the delivery device of the present invention may be applied to, but not limited to, various animals such as human, mouse, rat, rabbit, pig, dog and cat.
  • a method for administering to a test animal employs parenteral usage such as drip infusion, where conditions such as dosage, number of doses and duration of administration may appropriately be determined according to the type and the condition of the test animal.
  • the delivery device of the present invention may be used for a treatment (gene therapy) in which an intended nucleic acid is introduced into a cell responsible for any of various diseases.
  • the present invention may also provide a pharmaceutical composition comprising the polyplex for treating various diseases, a gene therapeutic agent comprising the pharmaceutical composition as an active ingredient for various diseases, and a method (particularly, a gene therapeutic method) employing the above-described PIC for treating various diseases.
  • cancers for example, lung cancer, pancreas cancer, brain tumor, hepatic cancer, breast cancer, colon cancer, neuroblastoma and bladder cancer
  • circulatory diseases for example, locomotor disorders and central nervous system diseases.
  • the above-described pharmaceutical composition may be prepared according to a conventional method by appropriately selecting an excipient, a filler, a bulking agent, a binder, a wetting agent, a disintegrating agent, a lubricant, a surfactant, a dispersant, a buffer, a preservative, a solubilizing aid, an antiseptic, a flavoring agent, a soothing agent, a stabilizer, a tonicity agent and the like generally used for drug production.
  • a form of the pharmaceutical composition employed is usually an intravenous injection (including drip) and provided, for example, in a unit dosage ampule or in multi-dosage vials.
  • a kit for delivering a nucleic acid of the present invention is characterized by comprising the above-described anionic polymer or a polymer complex of the present invention.
  • This kit may preferably be used for RNAi-based gene therapy in which siRNA is introduced into any target cell such as a cancer cell.
  • the storage state of the polymer is not limited and any state such as a solution or powder form may be selected considering the stability (storage property) and convenience.
  • the kit of the present invention may comprise components other than the above-described anionic polymer and polymer complex.
  • examples of other components include a nucleic acid to be introduced into a cell, various buffers for dissolution, dilution or the like, a lysis buffer, various proteins and an instruction (user manual), which may appropriately selected according to the intended use and the type of the polymer used.
  • the kit of the present invention is used for preparing a polyion complex (PIC) having, as a core moiety, a nucleic acid of interest (for example, plasmid DNA or siRNA) to be introduced into a target cell.
  • PIC polyion complex
  • the prepared PIC may effectively be used as a device for delivering a nucleic acid into a target cell.
  • N,N-dimethylformamide (DMF) Wako Pure Chemical Industries, Ltd, Japan
  • dichloromethane (DCM) Wako, Japan
  • n-butylamine ethylenediamine (1,2-diaminoethane), and diethylenetriamine (bis(2-aminoethyl)amine)
  • Acetic acid and hydrochloric acid were purchased and used without further purification (Wako, Japan).
  • 1-methyl-2-pyrrolidinone (NMP), cis-aconitic anhydride, succinic anhydride and bovine serum albumin were purchased from Sigma (St. Louis, MO).
  • ⁇ -benzyl-L-aspartate-N-carboxy-anhydride (BLA-NCA) was obtained from Nippon Oil and Fats Co., Ltd. (Tokyo, Japan).
  • PBLA was prepared by ring-opening polymerization of BLA-NCA initiated by the terminal amino group of n-butylamine.
  • n-butylamine (0.0417 mmol) was dissolved in 5 mL of DMF/DCM (1: 10).
  • the obtained polymer was precipitated in diethyl ether (150 mL). The crude precipitate was washed twice with diethyl ether to obtain the final product as white powder.
  • the degree of polymerization (DP) of BLA units was calculated to be 102.
  • JEOL EX 300 spectrometer was used to record the entire NMR spectra at 300MHz. Chemical shift downfield from tetramethylsilane is reported in ppm.
  • PBLA 0.802 mmol benzylester
  • NMP 10 mL
  • Diethylenetriamine (DET) (40.1 mmol) was added to the solution and the reaction mixture was stirred at 0°C for an hour.
  • the resulting solution was allowed to drip in 10% aqueous acetate solution (30 mL).
  • the neutralized solution was dialyzed at 4°C against 0.01M hydrochloric acid solution (x3) and distilled water (x3).
  • pAsp(DET) was obtained as white powder of hydrochloride salt. According to 1 H NMR, no benzyl peak was confirmed.
  • pAsp(DET) 0.055 mmol primary amine
  • 0.5 M NaHCO 3 buffer pH 9.0, 50 mL
  • Cis-aconitic anhydride 2.76 mmol
  • CAG promoter and a plasmid coding for luciferase were supplied from RIKEN Bioresource Center (Japan) ( H. Niwa et al. Gene, 1991, 108, 193-199 ).
  • SEYFP-F46L (Venus) fragment (which is a mutant of yellow fluorescent protein, containing mutation of F46L ( T. Nagai et al. Nat. Biotechnol., 2002, 20, 87-90 ) was supplied from RIKEN. This fragment was inserted into pCAcc vector (pCAcc+Venus).
  • This plasmid was amplified in competent DH5 E . coli, and purified using HiSpeed Plasmid MaxiKit (QIAGEN Science Co., Inc., Germany). The plasmid concentration was determined by absorbance at 260 nm.
  • pAsp(DET) (1 mg/mL) and plasmid DNA (50 ⁇ g/mL) were simply mixed at various N/P ratios (4-8) to obtain positive polyplexes.
  • the N/P ratios are defined as the excess molar ratios of amine units in pAsp(DET) to phosphate units in pDNA.
  • pAsp(DET-Aco) or pAsp(EDA-Suc) solution (1 mg/mL) was added to the positive polyplexes.
  • Each polyplex was diluted in an aqueous buffer (acetate buffer (pH 5.5, 10 mM) or Tris-HCl buffer (pH 7.4, 10 mM)). The final concentration of the plasmid DNA was 33 ⁇ g/mL.
  • Each sample was incubated at 37°C, and then subjected to dynamic light scattering (DLS) measurement using Zetasizer Nano-ZS (green badge, ZEN3500, Malvern, Ltd. Malvern, U.K.) with He-Ne ion laser at 633 nm.
  • DLS dynamic light scattering
  • Zeta potentials of the positive polyplex and the ternary polyplex were determined based on laser Doppler electrophoresis (detection angle of 173°, temperature at 37°C) using Zetasizer Nano-ZS (green badge, ZEN3500, Malvern, Ltd. Malvern, U.K.) with He-Ne ion laser at 633 nm. Each complex was diluted in an aqueous buffer (acetate buffer (pH 5.5, 10 mM) or Tris-HCl buffer (pH 7.4, 10 mM)). The final concentration of plasmid DNA was 33 ⁇ g/mL. Each sample was incubated at 37°C.
  • Human umbilical vein endothelial cells were seeded into a collagen-coated 24-well culture plate and incubated in 400 ⁇ l of EBM TM -2 containing insulin, hEGF, GA-1000, hFGF-B and FBS (5%), overnight. 40 ⁇ L of each sample solution was added to a medium (1 ⁇ g plasmid DNA/well). After 24 hours of incubation, the medium was exchanged for a fresh sample-free medium, and further incubated for 24 hours. The cells were washed with 400 ⁇ L of Dulbeccco's PBS and lysed with 100 ⁇ L of cell culture Promega lysis buffer.
  • the luciferase activities of the lysates were evaluated from the luminescence intensities with Mithras LB 940 (Berthold Technologies). The resulting luciferase was normalized according to the protein amount in the lysate determined with Micro BCA TM protein assay reagent kit (Pierce). Venus (YFP) expression was observed using Biozeero BZ-8000 (Keyence) at excitation wavelength of 450-490 nm (emission filter: 510-560 nm).
  • HUVEC was incubated with the sample for 24 hours, and viability was evaluated by MTT assay (Cell Counting Kit-9, Dojindo, Kumamoto, Japan). According to the protocol provided by the manufacturer, absorbance was read at 450 nm to measure each well. At the same time, the results were indicated as relative value (%) to the value of a control cell that has been incubated with Tris-HCl buffer (10 mM, pH 7.4) only.
  • BSA bovine serum albumin
  • HUVEC 30,000 cells were seeded into a 35 mm glass base dish (Iwaki, Japan) and incubated in 1mL of EBM TM -2 containing insulin, hEGF, GA-1000, hFGF-B and FBS (5%), overnight.
  • LSM 510 (Carl Zeiss, Germany) was used (with 63x object lens (C-Apochromat, Carl Zeiss, Germany)) for CLSM observation for LysoTraker, Cy5 and Hoechst 33342 at excitation wavelengths of 488 nm (Ar laser), 633 nm (He-Ne laser) and 710 nm (Mai Tai laser), respectively.
  • a polyplex of plasmid DNA and polycation was prepared.
  • the present inventors selected poly ⁇ N-[N'-(2-aminoethyl)-2-aminoethyl]aspartamide ⁇ (pAsp(DET)) (see Figure 2A ).
  • 1-4 equal molar amount of charge conversional polymer i.e., poly(N- ⁇ N'-[(N"-cis-aconityl)-2-aminoethyl]-2-aminoethyl ⁇ aspartamide) (pAsp(DET-Aco)) (see Figure 2A ), was added to the polyplex to form a ternary polyplex. Note that pAsp(DET-Aco) is altered to pAsp(DET)a that can efficiently disrupt the endosome at pH after degradation of cis-aconitic acid amide.
  • Each ternary polyplex showed unimodal size distribution at various charge ratios and had an average diameter of about 130 nm according to dynamic light scattering (DLS) measurement even in the presence of excessive pAsp(DET-Aco).
  • DLS dynamic light scattering
  • the charge conversional behavior of the ternary polyplex was monitored from the change in the zeta potential shown in Figure 2B .
  • the ternary polyplex maintained a zeta potential of around -40 mV at pH7.4.
  • the zeta potential at pH 5.5 gradually changed from negative to positive, demonstrating charge conversion resulting from degradation of the cis-aconitic acid amide moiety.
  • the zeta potential reached 0 mV.
  • the present inventors used a non-charge-conversional polycation having a similar structure, i.e., poly[(N'-succinyl-2-aminoethyl)aspartamide] (pAsp(EDA-Suc)) (see Figure 2A ).
  • pAsp(EDA-Suc) poly[(N'-succinyl-2-aminoethyl)aspartamide]
  • Figure 2A A ternary polyplex of pAsp(DET) and pAsp(EDA-Suc) maintained a zeta potential of around -40 mV at pH 5.5 and pH 7.4, with no sign of charge conversion (see "Materials and Methods" and Figure 7 ).
  • Transfection was carried out using human umbilical vein endothelial cell (HUVEC). Only limited transfection reagents can be used since transfection of HUVEC is very difficult and HUVEC is highly sensitive to toxicity [10] .
  • HUVEC human umbilical vein endothelial cell
  • the ternary polyplex of pAsp(EDA-Suc) as a control showed similar transfection efficiency as that of the primary polyplex of pAsp(DET), the charge conversional ternary polyplex of pAsp(DET-Aco) showed transfection efficiency that was more than ten times the transfection efficiency of ExGen 500 (linear PEI of a commercially available transfection reagent) and twice as high as the transfection efficiency of the pAsp(DET) polyplex.
  • the negative surface charge of the ternary complex is useless for cellular uptake or endosomal escape, it is capable of increasing the stability and reducing the toxicity of the complex in the presence of the serum protein as shown in Figure 3A .
  • a non-charge-conversional ternary polyplex (DNA/pAsp(DET)/pAsp(DET-Suc)) was also capable of showing similar transfection efficiency.
  • the charge conversional endosome disruption moiety was introduced into the ternary polyplex (DNA/pAsp(DET)/pAsp(DET-Aco)) in favor of stability and low toxicity, transfection efficiency was further increased.
  • the results of transfection using yellow fluorescent protein (YFP) pDNA are summarized in "Materials and Methods" described below (see Figures 8 and 9 ), which also show appropriate transfection efficiency of the ternary polyplex system.
  • Cytotoxicity measured by MTT assay is shown in Figure 3C .
  • ExGen 500 showed very high toxicity with viability of less than 10 %.
  • the viability of pAsp(DET) polyplex also decreased to 50%.
  • One of the main reasons of the decreased viability was probably the membrane toxicity induced by the positive surface charge of the polyplex [11] .
  • Cy5-labeled pDNA was used to examine the intracellular distribution of the polyplex with a confocal laser microscope (CLSM) (see Figure 4 ). Upon release of the polyplex from acidic organelle, the yellow fluorescence changes to red. The positively-charged pAsp(DET) polyplex showed significant endosomal escape even after 3 hours, and over 80% of DNA escape was observed after 24 hours. After 3 hours, both ternary polyplexes showed low endosomal escape.
  • CLSM confocal laser microscope
  • the present inventors developed a ternary polyplex that was negatively charged at pH outside a cell and the charge changes to positive at pH in an endosome, where the endosome is disrupted.
  • the present inventors realized fairly high transfection activity and low toxicity effect on highly sensitive primary cells (HUVEC).
  • the transfection efficiency of this ternary polyplex system may be further enhanced via conjugation with an appropriate ligand (for example, an RGD peptide for activating internalization through binding to integrin receptor) [12] .
  • an appropriate ligand for example, an RGD peptide for activating internalization through binding to integrin receptor
  • the efficient and chemical toxicity-free transfection according to this concept is one of the most important and urgent issues in the biomedical field.
  • stability of the ternary polyplex in the presence of a negatively-charged serum protein is also advantageous for development of an in vivo gene vector.
  • PAsp(DET(aco)) or PAsp(EDA(suc)) was added and left to stand at 4°C for 3 hours to prepare a ternary polyplex.
  • 20,000 cells per well were seeded into a 24-well dish, cultured for 24 hours and added with 31 ⁇ l of polyplex (pDNA concentration: 2/3 OD). The cell and the polyplex were brought into contact with each other for 24 hours. Then, the medium (DMEM) was exchanged for another 24 hours of cultivation. The gene expression activity was assessed by luciferase assay.
  • Human hepatic cancer cell Huh-7
  • normal human umbilical vein endothelial cell Huh-6
  • Cells (Huh-7) were seeded into 35 mm petri dishes at 50,000 cells/well, cultured for 24 hours and added with 90 ⁇ l of polyplex prepared with fluorescent dye (Cy5)-labeled pDNA (pDNA concentration: 2/3 OD). The cells were brought into contact with the polyplex for 24 hours, and then the nuclei and the endosomes were stained with 5 ⁇ M of Lysto Tracker (20 ⁇ L) and Hoechst (10 ⁇ L). Just before the measurement, washing was performed with PBS for three times and 2 ml of medium was added for observation. LSM-510 from Carl Zeiss was used as the confocal laser microscope.
  • Deprotected polymer samples were also similarly subjected to analyses by 1 H NMR and GPC.
  • PAsp[DET(aco)] and PAsp[EDA(suc)] were synthesized in the same manner as described in Example 1.
  • PAsp(EDA(suc))-added ternary polyplex was equivalent to or lower than that of non-added cationic pDNA/PLL.
  • the gene expression activity of PAsp[DET(aco)]-added ternary polyplex increased as compared to that of pDNA/PLL by about 20 and 10 times for Huh-7 and HUVEC, respectively ( Figure 12 ). Since PAsp[DET(aco)] polymer has a charge conversion function where its charge inverts in response to pH, this property seems to contribute to the increase in the expression activity.
  • the polyplexes trapped in the endosomes were quantitatively evaluated based on these confocal laser microscope images.
  • 70% or more of the cationic polyplex pDNA/PLL and the ternary polyplex added with PAsp[EDA(suc)] remained trapped in the endosome ( Figure 15 ).
  • a polyplex using PLL homopolymer and a derivative thereof has very poor property of escaping from the endosome.
  • PAsp[EDA(suc)] does not have a function of inverting the charge in response to acidic pH, even when it is made into a ternary polyplex, improvement of the intracellular dynamics, that is, enhancement of the efficiency of escaping from the endosome, was not realized.
  • N-succinimidyl octadecanoate was synthesized according to a known method [ N. M. Howarth, W. E. Lindsell, E. Murray, P. N. Preson, Tetrahedron 61 (2005) 8875-8887 ].
  • Stearic acid (1.87 g, 6.56 mmol) and N-hydroxysuccinimide (0.76 g,6.56 mmol) was dissolved in 80 mL of dichloromethane (DCM), and allowed to react with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (WSC) (1.25 g, 6.56 mmol) for 48 hours.
  • DCM dichloromethane
  • WSC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • poly(L-Lysine) (molecular weight 20,000) and DIPEA were dissolved in methanol, and allowed to react with N-succinimidyl octadecanoate dissolved in a small amount of dichloromethane at 4°C for 24 hours. At the end of the reaction, the resultant was reprecipitated with diethylether. The filtrated sample was dissolved in methanol/water (1: 1 v/v) and dialyzed three times against 0.01M aqueous HCl solution and once against distilled water at 4°C. Subsequently, the resultant was collected by lyophilization, thereby synthesizing poly(L-Lysine) introduced with stearoyl groups into the side-chain for 15%, 28% and 45% (PLL-ST).
  • siRNA/PLL-ST ternary polyplex was obtained by mixing siRNA dissolved in HEPES buffer (10mM, pH 7.4) with PLL-ST such that the ratio of the phosphate residue of siRNA to the lysine residue was 1.4, then mixing the resultant with PAsp(DET(aco)) at different mixture ratios, and left to stand at 4°C for 3 hours.
  • Huh-7 cell was seeded at 40,000 cells/well, transfected with plasmids that express GL3 luciferase and Rellina luciferase using Lipofectamine 2000, added with GL3 luciferase siRNA-bearing ternary polyplex (siRNA concentration: 100nM) and cultured for 48 hours.
  • siRNA concentration: 100nM GL3 luciferase siRNA-bearing ternary polyplex
  • the inhibitory effect of siRNA on the expression of GL3 luciferase was evaluated by calculating the ratio of luciferase luminescence (GL3/Rellina) after the addition of GL3 and Rellina substrates.
  • Huh-7 cell was seeded at 10,000 cells/well, added with GL3 luciferase siRNA-bearing ternary polyplex (siRNA concentration: 100nM) and cultured for 48 hours. Thereafter, viability of the cell was evaluated by MTT assay.
  • the present invention provides a charge conversional ternary polyplex.
  • the polyplex of the present invention is capable of delivering a nucleic acid to a cell with high efficiency without raising toxicity, and thus is extremely useful for gene therapy and the like.

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EP2462938A1 (fr) * 2009-07-23 2012-06-13 The University of Tokyo Polymère anionique, complexe polyions utilisant un polymère anionique, composite de polymère ternaire, et composition pharmaceutique
WO2014169256A3 (fr) * 2013-04-11 2014-12-11 Vanderbilt University Polyplexes
US9051437B2 (en) 2010-07-28 2015-06-09 The University Of Tokyo Electrostatically bonded vesicle

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US20090232762A1 (en) * 2008-03-11 2009-09-17 May Pang Xiong Compositions for delivery of therapeutic agents
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JP4655298B1 (ja) 2010-02-23 2011-03-23 ナノキャリア株式会社 短鎖のカチオン性ポリアミノ酸およびその使用
EP2640400A4 (fr) * 2010-11-19 2016-01-20 Sirna Therapeutics Inc Polymères poly(amide) destinés à l'administration d'oligonucléotides
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US20150141575A1 (en) 2015-05-21
US20110060123A1 (en) 2011-03-10
JPWO2009133968A1 (ja) 2011-09-01
US9303122B2 (en) 2016-04-05
EP2284210B1 (fr) 2017-12-06
EP2284210A4 (fr) 2013-08-07
JP5645186B2 (ja) 2014-12-24

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